The present invention relates generally to the chemical preparation of purine nucleosides. More specifically, the invention relates to the coupling of an adenine derivative with a blocked arabinofuranosyl to form a xcex2-D-adenine nucleoside. Such nucleosides are valuable compounds in the field of cancer therapy and as anti-viral agents.
A number of xcex2-D-purine nucleosides derived from adenine are useful as antitumor and antiviral agents. An important step in the synthesis of such agents is the formation of the N-glycoside bond between the adenine nucleobase and an arabinofuranosyl derivative. The coupling reactions used to form the N-glycoside bond of 2xe2x80x2-deoxynucleosides have typically resulted in the formation of a mixture of xcex1 and xcex2-anomers.
Nucleosides have been synthesized by fusion glycosylation, wherein the reaction is carried out in the absence of solvent at a temperature sufficient to convert the reactants to a molten phase. E.g., 2,6-dichloropurine has been coupled under fusion conditions with 5-O-benzyl-2-deoxy-1,3-di-O-acetyl-2-fluroarabinose to form a 2xe2x80x2-fluoroarabinonucleoside in 27% yield (Wright et al., J. Org. Chem. 34:2632, 1969). Another synthetic method utilizes silylated nucleobase derivatives, e.g., a silylated nucleobase has been coupled with a peracetylated deoxy-sugar in the presence of a solvent and a Friedel Crafts catalyst (Vorbruggen et al., J. Org. Chem. 41:, 2084, 1976). This method has been modified by incorporating a sulfonate leaving group in the deoxy-sugar in the synthesis of 2xe2x80x2-deoxy-2xe2x80x2-difluoronucleosides (U.S. Pat. No. 4,526,988; U.S. Pat. No. 4,965,374).
High yields of 2xe2x80x2-deoxy-2xe2x80x2-fluoro-pyrimidine nucleosides were obtained from refluxing pyrimidines with 2-deoxy-2-fluoro-3,5-di-O-benzoyl-xcex1-O-arabinofuranosyl bromide. (Howell et al., J. Org. Chem. 53:85-88, 1988). It was found that use of solvents with lower dielectric constants produced have higher xcex2:xcex1 anomer ratios. It was postulated that such solvents favored an SN2 reaction, whereas solvents with higher dielectric constants favored production of xcex1-anomers via an ionic SN1 pathway.
Anion glycosylation procedures have also been used to prepare 2xe2x80x2-deoxy-2xe2x80x2-fluoropurine nucleosides. EP 428109 discloses the coupling of the sodium salt of 6-chloropurine, formed by sodium hydride, with 3,5-dibenzyl-xcex1-D-arabinofuranosyl bromide using conditions that favor SN2 displacement. Use of 1:1 acetonitrile/methylene chloride resulted in a nucleoside product with a xcex2:xcex1 anomer ratio 10:1, as opposed to a ratio of 3.4:1 observed when using a silylated purine reactant. In regard to the use of adenine salts, the amino substituent at the C-6 position was protected as a benzoyl derivative during the coupling reaction. Protecting the exocyclic amino group precludes the formation of arabinofuranosyl adducts which otherwise may be expected to be produced (e.g., Ubukata et al., Tetrahedron Lett., 27:3907-3908, 1986; Ubukata et al., Agric. Biol. Chem., 52: 1117-1122, 1988; Searle et al., J. Org. Chem., 60:4296-4298, 1995; Baraldi et al., J. Med. Chem., 41:3174-3185, 1998). The preparation of xcex1 and xcex2 anomers of 2xe2x80x2-deoxy-2xe2x80x2-fluoropurine and 2xe2x80x2-difluoropurine nucleosides by anion glycosylation are disclosed by U.S. Pat. No. 5,744,597 and U.S. Pat. No. 5,281,357, with xcex2-anomer enriched nucleosides prepared in a xcex2:xcex1 anomer ratio of greater than 1:1 to about 10:1 and from greater that 1:1 to about 7:1 respectively. In regard to purines substituted with exocyclic amino groups, both patents again disclose protecting such groups during coupling to an appropriate sugar moiety. U.S. Pat. No. 5,281,357 also discloses the effect of solvents on the xcex2:xcex1 anomer ratio of 9-[1-(2xe2x80x2-deoxy-2xe2x80x2, 2xe2x80x2-difluoro-3xe2x80x2,5xe2x80x2-di-O-benzoyl-D-ribofuranosyl)]-2,6-dipivalamidopurine prepared by coupling the potassium salt of 2,6-dipivalamidopurine with an xcex1 anomer enriched preparation of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-1-trifluoromethanesulfonate. There was no correlation between the dielectric constant of the six solvents used and the xcex2:xcex1 anomer ratio, e.g. ethyl acetate and acetonitrile both gave the same ratio of 1.6:1. t-Butyl alcohol gave the highest xcex2:xcex1 anomer ratio of 3.5:1.
Despite the preparative methods for purine nucleosides known in the art, there is still a need for economically preferable, effective and efficient process for the preparation of these compounds. The object of the present invention is to provide such a process. Further objects are to minimize the number of process reaction steps and to provide a process that is readily scalable for the production of commercial-scale quantities. Other objects and advantages will become apparent to persons skilled in the art and familiar with the background references from a careful reading of this specification.
In its most general terms, one aspect of the present invention provides for the preparation of xcex2-adenine nucleosides by coupling an adenine derivative containing an unprotected exocyclic amino group at the C-6 position, and a blocked arabinofuranosyl derivative. In preferred embodiments, this reaction can be depicted as: 
R1 is hydrogen, halogen or xe2x80x94OR6, wherein R6 is a hydroxy protecting group. In a preferred embodiment R1 is fluoro. R2 and R3 are hydroxy-protecting groups. In preferred embodiments R2, R3 and R6 are independently benzoyl or acetyl. R4 is a leaving group. Suitable leaving groups include, halo, fluorosulfonyl, alkylsulfonyloxy, trifluoroalkylsulfonyloxy and arylsulfonyloxy. In a preferred embodiment, R4 is bromo. R5 is hydrogen, halogen or xe2x80x94NH2. In preferred embodiments, R5 is chloro or fluoro.
Surprisingly, this reaction proceeds without substantial production of adducts resulting from addition of the blocked arabinofuranosyl (1) with the exocyclic amino group at the C-6 position of compound (2) (hereinafter termed xe2x80x9cC-6 exocyclic amino groupxe2x80x9d), which remains unprotected during the reaction, and/or the nitrogen at the N-7 position of the adenine ring. An example of an undesired C-6 exocyclic amino group by-product adduct is represented by the following formula: 
For the purposes of the present invention, and in light of the objective to provide an economically preferable, effective and efficient process, xe2x80x9csubstantial formationxe2x80x9d means conversion of about 40% of the adenine derivative of formula (2) to a by-product adduct or adducts resulting from addition of the blocked arabinofuranosyl of formula (1) to the unprotected C-6 exocyclic amino group and/or N-7 position of compound (2). In embodiments wherein R5 is xe2x80x94NH2 (hereinafter termed xe2x80x9cR5 xe2x80x94NH2 groupxe2x80x9d), xe2x80x9csubstantial formationxe2x80x9d means conversion of about 40% of the adenine derivative of formula (2) to by-product adduct(s) resulting from addition of the blocked arabinofuranosyl of formula (1) to the unprotected C-6 exocyclic amino group and/or N-7 position and/or the R5 xe2x80x94NH2 group of compound (2).
Even more surprising is that the reaction can proceed without even a significant production of adducts resulting from addition of the blocked arabinofuranosyl (1) with the C-6 exocyclic amino group and/or N-7 position of compound (2). For the purposes of the present invention, xe2x80x9csignificant productionxe2x80x9d means conversion of about 5% of the adenine derivative of formula (2) to a by-product adduct or adducts resulting from addition of the blocked arabinofuranosyl (1) to the unprotected C-6 exocyclic amino group and/or N-7 position of compound (2). In embodiments wherein R5 is xe2x80x94NH2, xe2x80x9csignificant productionxe2x80x9d means conversion of about 5% of the adenine derivative of formula (2) to a by-product adduct(s) resulting from addition of the blocked arabinofuranosyl of formula (1) to the unprotected C-6 exocyclic amino group and/or N-7 position and/or the R5 xe2x80x94NH2 group of compound (2).
Useful bases are generally those with a pKa in water of 15 or greater. In preferred embodiments, the base is an alkali metal base, more preferred being a potassium base. In preferred embodiments, the base is a sterically hindered base, e.g., potassium t-butoxide or potassium t-amylate. Suitable inert solvents include, but are not limited to, t-butyl alcohol, acetonitrile, dichloromethane, dichloroethane, t-amyl alcohol, tetrahydrofuran or mixtures thereof. In preferred embodiments, the solvent or solvent mixture has a boiling point of about 80xc2x0 C. or greater.
The process of the present invention also further comprises de-protection of the blocked carbohydrate moiety to form a xcex2-nucleoside of the formula: 
wherein, R1 and R5 are as defined above.
In some embodiments, the adenine derivative is 2-chloroadenine and the blocked arabinofuranosyl derivative is a 2-deoxy-2-fluoro-arabinofuranosyl derivative, whereupon the resulting xcex2-nucleoside is a 2-chloro-9-(2xe2x80x2-deoxy-2xe2x80x2-fluoro-xcex2-D-arabinofuranosyl) adenine derivative. The reaction can be depicted as: 
wherein R 2, R3 and R4 are as defined above. The process also further comprises de-protecting the carbohydrate moiety to form 2-chloro-9-(2xe2x80x2-deoxy-2xe2x80x2-fluoro-xcex2-D-arabinofuranosyl) adenine, also known as clofarabine.
Another aspect of the invention is the discovery of the surprising steroselectivity that can be achieved in the production 2xe2x80x2-deoxy-2xe2x80x2-halo-xcex2-D-adenine nucleosides wherein such nucleosides are also produced in high yield. This reaction can be depicted as: 
R7 and R8 are independently halogen, M+is potassium, and R2, R3, and R5 are as defined above. Halogen includes bromo, fluoro, chloro and iodo. In a preferred embodiment R8 is fluoro. In various embodiments R7 is chloro or, preferably, bromo. In some embodiments, the process further comprises the addition of calcium hydride. Suitable inert solvents include t-butyl alcohol, acetonitrile, dichloromethane, dichloroethane, t-amyl alcohol, tetrahydrofuran or mixtures thereof. In preferred embodiments, the solvent is a mixture of t-butyl alcohol and acetonitrile, or a mixture of t-butyl alcohol and dichloroethane, or a mixture of dichloroethane and acetonitrile, or a mixture of t-amyl alcohol and dichloroethane, or a mixture of t-amyl alcohol and acetonitrile, or a mixture of t-amyl alcohol, acetonitrile and dichloromethane, or a mixture of t-amyl alcohol, acetonitrile and dichloroethane. In preferred embodiments, the solvent or solvent mixture has a boiling point of about 80xc2x0 C. or greater.
In some embodiments, the adenine derivative salt (10) is formed in situ by the reaction of a potassium base with the corresponding adenine derivative (2). In preferred embodiments, the base is potassium t-butoxide or potassium t-amylate.
In various embodiments of the invention, the coupling reaction produces a preparation wherein the ratio of the xcex2-anomer of formula (11) to the xcex1-anomer of formula (12) is at least about 10:1, or preferably is at least about 15:1, or more preferable is at least about 20:1. Thus, the anomer ratio may be 10:1 or greater, 15:1 or greater or 20:1 or greater. In preferred embodiments the xcex2-anomer of formula (11) is prepared in a yield of about 40% or greater. In more preferred embodiments, the xcex2-anomer of formula (11) is prepared in yields of about 50% or greater or about 80% or greater.
The process of the present invention may also further comprises isolation of the xcex2-anomer (11) by subjecting the mixture of xcex2 and xcex1-anomers to recrystallization or by a re-slurry procedure. In a preferred embodiment, the further purification comprises reslurry from methanol or crystallization from a mixture of butyl acetate and heptane. In various embodiments, the purified preparation comprises a mixture of nucleosides wherein the ratio of the xcex2-anomer of formula (11) to the xcex1-anomer of formula (12) is at least about 20:1, or least about 40:1, or at least about 60:1.
The process also further comprises de-protection of the blocked carbohydrate moiety of the protected xcex2-anomer to form a xcex2-nucleoside of the formula: 
wherein, R5 and R8 are as defined above. When R5 is chloro and R8 is fluoro, the unblocked xcex2-nucleoside of formula (13) is 2-chloro-9-(2xe2x80x2-deoxy-2xe2x80x2-fluoro-xcex2-D-arabinofuranosyl) adenine.
Another aspect of the present invention is a multi-step process for the preparation of a composition comprising 2-chloro-9-(2xe2x80x2-deoxy-2xe2x80x2-fluoro-xcex2-D-arabinofuranosyl) adenine. This comprises the integration of the other aspects of the present invention into an economically preferable, effective and efficient synthesis and isolation of 2-chloro-9-(2xe2x80x2-deoxy-2xe2x80x2-fluoro-xcex2-D-arabinofuranosyl) adenine. This process minimizes the number of steps in part by not requiring protection of the C-6 exocyclic amino group. In addition, the surprising stereoselective preference for the xcex2-anomer in part enables the preparation of a composition with an xcex2:xcex1 anomer ratio of at least 99:1 or in preferred embodiments is about 400:1 or greater, about 500:1 or greater or about 1000:1 or greater, without utilizing a preparative chromatography step for the purification of the xcex2-anomer. The absence of a chromatographic step is a major advantage in regard to an economically preferable commercial-scale process.
The process comprises reacting 3,5-O-dibenzoyl-2-deoxy-2-fluoro-xcex1-D-arabinofuranosyl bromide with a 2-chloroadenine potassium salt of the formula: 
in the presence of a solvent to form 2-chloro-9-(3xe2x80x2,5xe2x80x2-O-dibenzoyl-2xe2x80x2-deoxy-2xe2x80x2-fluoro-xcex2-D-arabinofuranosyl) adenine. The C-6 exocyclic amino group of the 2-chloroadenine potassium salt is not protected during the process. The 2-chloro-9-(3xe2x80x2,5xe2x80x2-O-dibenzoyl-2xe2x80x2-deoxy-2xe2x80x2-fluoro-xcex2-D-arabinofuranosyl) adenine is then de-protected to form 2-chloro-9-(2xe2x80x2-deoxy-2xe2x80x2-fluoro-xcex2-D-arabinofuranosyl) adenine, which is then isolated to provide a composition comprising 2-chloro-9-(2xe2x80x2-deoxy-2xe2x80x2-fluoro-xcex2-D-arabinofuranosyl) adenine. In some embodiments, wherein the composition produced by the multi-step process, as described above, also comprises 2-chloro-9-(2xe2x80x2-deoxy-2xe2x80x2-fluoro-xcex2-D-arabinofuranosyl) adenine, the 2-chloro-9-(2xe2x80x2-deoxy-2xe2x80x2-fluoro-xcex2-D-arabinofuranosyl) adenine is substantially pure. For the purposes of the present invention, substantially pure 2-chloro-9-(2xe2x80x2-deoxy-2xe2x80x2-fluoro-xcex2-D-arabinofuranosyl) adenine means that the ratio of xcex2-anomer to xcex1-anomer as measured by high pressure liquid chromatography and spectrophotometric analysis, is at least 99:1.
The process may further comprise isolating the 2-chloro-9-(3xe2x80x2,5xe2x80x2-O-dibenzoyl-2xe2x80x2-deoxy-2xe2x80x2-fluoro-xcex2-D-arabinofuranosyl) adenine before the deprotection step. In some embodiments, this isolation may comprise reslurry and/or recrystallization, which may be effected by use of methanol or by use of a mixture of butyl acetate and heptane. In other embodiments, the isolation of 2-chloro-9-(2xe2x80x2-deoxy-2xe2x80x2-fluoro-xcex2-D-arabinofuranosyl) adenine also comprises recrystallization. In some embodiments, the recrystallization is from methanol.
In some embodiments, the 2-chloroadenine potassium salt is prepared in situ by the reaction of a potassium base with 2-chloroadenine in a suitable inert solvent. In preferred embodiments, the base is potassium t-butoxide or potassium t-amylate. Suitable inert solvents include t-butyl alcohol, acetonitrile, dichloromethane, dichloroethane, t-amyl alcohol, tetrahydrofuran or mixtures thereof. In preferred embodiments, the solvent is a mixture of t-butyl alcohol and acetonitrile, or a mixture of t-butyl alcohol and dichloroethane, or a mixture of dichloroethane and acetonitrile, or a mixture of t-amyl alcohol and dichloroethane, or a mixture of t-amyl alcohol and acetonitrile, or a mixture of t-amyl alcohol, acetonitrile and dichloromethane, or a mixture of t-amyl alcohol, acetonitrile and dichloroethane.